Polypeptides against IL-23

ABSTRACT

The present invention relates to amino acid sequences that are directed against Interleukin-23 (IL-23). The amino acid sequences of the present invention comprise two NANOBODIES® against IL-23 and one NANOBODY® against serum albumin, linked by two linkers (9GS linkers). In particular, the invention relates to the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 (listed in Table 1 and FIG. 1) (also referred to herein as “anti-IL 23 polypeptides of the invention”).

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofInternational Patent Application Serial No. PCT/EP2016/076076, filedOct. 28, 2016, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application Ser. No. 62/248,468, filed Oct. 30, 2015, thecontents of each of which is incorporated by reference herein in itsentirety for all purposes.

INCORPORATION BY REFERENCE

This application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated herein byreference in its entirety. This ASCII copy, created on Apr. 20, 2018, isnamed A084870183US01-SEQ-JRV and is 78.375 kB in size.

The present invention relates to amino acid sequences that are directedagainst IL-23.

The amino acid sequences of the present invention comprise twoNANOBODIES® (immunoglobulin single variable domains) against IL-23 andone NANOBODY® (immunoglobulin single variable domain) against serumalbumin, linked by two linkers (9GS linkers).

In particular, the invention relates to the amino acid sequences of SEQID NO: 2 and SEQ ID NO: 3 (listed in Table 1 and FIG. 1) (also referredto herein as “anti-IL 23 polypeptides of the invention”).

TABLE 1 Polypeptides of the invention SEQ IDDVQLLESGGGVVQPGGSLRLSCAASGRIFSLPASGNIFNLL NO: 2TIAWYRQAPGKGRELVATINSGSRTYYADSVKGRFTISRDNSKKTVYLQMNSLRPEDTALYYCQTSGSGSPNFWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLLESGGGVVQPGGSLRLSCAASGRTLSSYAMGWFRQAPGKGREFVARISQGGTAIYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCAKDPSPYYRGSAYLL SGSYDSWGQGTLVTVSSA SEQ IDDVQLLESGGGVVQPGGSLRLSCAASGRIFSLPASGNIFNLL NO: 3TIAWYRQAPGKGRELVATINSGSRTYYADSVKGRFTISRDNSKKTVYLQMNSLRPEDTALYYCQTSGSGSPNFWGQGTLVKVSSGGGGSGGGSEVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVKVSSGGGGSGGGSEVQLLESGGGVVQPGGSLRLSCAASGRTLSSYAMGWFRQAPGKGREFVARISQGGTAIYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCAKDPSPYYRGSAYLL SGSYDSWGQGTLVKVSSA

Other aspects, embodiments, features, uses and advantages of theinvention will be clear to the skilled person based on the disclosureherein.

In the present application, the amino acid residues/positions in animmunoglobulin heavy-chain variable domain will be indicated with thenumbering according to Kabat. For the sake of convenience, FIG. 2 givesa table listing some of the amino acid positions that will bespecifically referred to herein and their numbering according to somealternative numbering systems (such as Aho and IMGT. Note: unlessexplicitly indicated otherwise, for the present description and claims,Kabat numbering is decisive; other numbering systems are given forreference only).

Amino acid sequences against IL-23 based on related NANOBODY® buildingblocks are known from WO 2009/068627, WO 2010/142534 and WO2011/135026.These prior art sequences are given in Table 1 as SEQ ID NO's: 4 to 23.FIG. 3 shows an alignment of these prior art sequences with thesequences of SEQ ID NO: 2 and SEQ ID NO: 3 according to the invention(the sequence of SEQ ID NO:1 is a reference sequence that has beenincluded for the purpose of making the alignment, i.e. in order toclearly show the amino acid “differences” between the amino acidsequences of the invention and the prior art sequences of SEQ ID NO:s 4to 23. SEQ ID NO: 1 corresponds to the prior art sequence of SEQ IDNO:22, but with an N-terminal Asp-residue (D) instead of an N-terminalGlu-residue (E). This reference sequence is also used as a comparator inExample 1.

Compared to the amino acid sequences of SEQ ID NOs: 4 to 23, the anti-IL23 polypeptides of the invention contain a number of specific amino acidresidues/mutations (shown in the alignment of FIG. 3), as well as aC-terminal alanine residue. As a result of the presence of thesemutations and alanine-extension, the amino acid residues of theinvention show much reduced binding by so-called “pre-existingantibodies” (for which reference is made WO 12/175741, WO 2013/024059and also for example by Holland et al. (J. Clin. Immunol. 2013,33(7):1192-203) as well as to the co-pending non-prepublished PCTapplication PCT/EP2015/060643 by Assignee filed on May 13, 2015 andentitled “Improved immunoglobulin variable domains” (published on Nov.19, 2015 as WO 2015/173325) compared to the prior art sequences of SEQID NO's 4 to 23.

All terms not explicitly defined herein are as defined in WO2009/068627.

In a first aspect, the invention relates to an amino acid sequence thatis chosen from the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO:3. These amino acid sequences can bind to (and can in particularspecifically bind to, as defined in WO 2009/068627) IL-23 and/or canmodulate signaling mediated by IL-23 (or its receptor) and they can beused to prevent or treat diseases associated with IL-23 (as describedherein and in WO 2009/068627, WO 2010/142534 and WO2011/135026).

In a specific aspect, the invention relates to an amino acid sequencethat is the amino acid sequence of SEQ ID NO: 2.

In yet another specific aspect, the invention relates to an amino acidsequence that is the amino acid sequence of SEQ ID NO: 3.

It will be clear to the skilled person from the disclosure herein thatthe anti-IL-23 polypeptides of the invention are directed against (asdefined WO 2009/068627) IL-23 and are improved variants of the prior artsequences referred to herein. Thus, the anti-IL-23 polypeptides of theinvention can be used for the same purposes, uses and applications asthe prior art sequences, for example to modulate signaling that ismediated by IL-23 and/or its receptor(s); and/or in the prevention ortreatment of diseases associated with IL-23 and/or with signaling thatis mediated by IL-23, such as for example inflammation and inflammatorydisorders such as bowel diseases (ulcerative colitis, Crohn's disease,IBD), infectious diseases, psoriasis, cancer, autoimmune diseases (suchas MS), sarcoidosis, transplant rejection, cystic fibrosis, asthma,chronic obstructive pulmonary disease, rheumatoid arthritis, viralinfection, common variable immunodeficiency, and the various diseasesand disorders mentioned in the prior art cited herein. Further referenceis again made to WO 2009/068627, WO 2010/142534 and WO2011/135026.

For example, as mentioned on pages 4-5 of WO 09/068627, IL23 was shownto be responsible for the chronic inflammation observed in inflammatorybowel disease. This was confirmed by the fact that the IL23R gene wasidentified as being involved in inflammatory bowel disease. It has alsobeen found that p19 knock out mice are resistant to collagen-inducedarthritis and colitis, whereas comparable p35 knock out mice were foundto be more susceptible to collagen-induced arthritis. Also, when p19knock out mice were crossed with IL-10 knock out mice, the resultingoffspring were resistant to colitis, whereas similar crosses of p19knock out mice with IL-10 knock out mice resulted in offspring that wassusceptible to colitis. It was further found that a monoclonal antibodyagainst p19 inhibits the development of EAE, a preclinical animal modelfor multiple sclerosis, and reduces serum levels of IL-17 (which is notregulated by IL-12). Also, IL-23 rather than IL-12 appears to be theessential cytokine in CNS autoimmune inflammation. All this resultssuggests that IL-23/p19 may be an attractive target for the treatment ofcolitis, Crohn's diseases, IBD, multiple sclerosis, rheumatoid arthritisand some of the other diseases and disorders mentioned herein. Also,IL23 and IL27—two of the other heterodimeric cytokines from the IL-12family—also regulate TH1-cell response, albeit with distinct functions.The ability of IL-23 to stimulate CD4+ T cells to produce IL-17 also hasbeen described as having a dominant role in the development andmaintenance of autoimmune inflammation.

Also, Example 45 of WO 09/068627 shows that the polypeptides of WO09/068627 (and thus, by extension, the anti-IL-23 polypeptides of theinvention) can also be valuable in the prevention and treatment ofpsoriasis (either by systemic/parenteral administration or by topicaltreatment, e.g. using a créme or lotion (see page 328 and 331-332 of WO09/068627).

In another aspect, the invention relates to a nucleic acid that encodesan anti-IL-23 polypeptide of the invention. Such a nucleic acid willalso be referred to herein as a “nucleic acid of the invention”.

In another aspect, the invention relates to a host or host cell thatexpresses (or that under suitable circumstances is capable ofexpressing) an anti-IL-23 polypeptide of the invention; and/or thatcontains a nucleic acid of the invention. Such a host or host cell mayagain generally be as described in WO 09/068627 (see for example pages315-328).

The invention also relates to methods for the production/expression ofan anti-IL-23 polypeptide of the invention. Such methods may generallycomprise the steps of (i) the expression, in a suitable host cell orhost organism or in another suitable expression system of a nucleic acidthat encodes an anti-IL-23 polypeptide of the invention, optionallyfollowed by: (ii) isolating and/or purifying the anti-IL-23 polypeptideof the invention thus obtained. In particular, such a method maycomprise the steps of (i) cultivating and/or maintaining a host of theinvention under conditions that are such that said host of the inventionexpresses and/or produces an anti-IL-23 polypeptide of the invention;optionally followed by (ii) isolating and/or purifying the anti-IL-23polypeptide of the invention thus obtained. These methods again mayessentially be performed as described in WO 09/068627 (see for examplepages 315-328).

One specific method for the production/expression of the anti-IL-23polypeptides of the invention is described in the Internationalapplication of Ablynx N. V. entitled “Method for the production ofdomain antibodies”, which has an international filing date of Apr. 30,2010.

The invention further relates to a product or composition containing orcomprising an anti-IL-23 polypeptide of the invention and/or at leastone nucleic acid of the invention, and optionally one or more furthercomponents of such compositions known per se, i.e. depending on theintended use of the composition. Such a product or composition may forexample be a pharmaceutical composition (as described herein), aveterinary composition or a product or composition for diagnostic use(as also described herein). Such products or compositions may againgenerally be as described in WO 09/068627 (see for example pages329-337).

The invention also relates to the use of an anti-IL-23 polypeptide ofthe invention, or of a composition comprising the same, in (methods orcompositions for) modulating (as defined in WO 09/068627) IL-23 and/orIL-23-mediated signaling (as defined in WO 09/068627), either in vitro(e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a singlecell or in a multicellular organism, and in particular in a mammal, andmore in particular in a human being, such as in a human being that is atrisk of or suffers from a disease or disorder associated withheterodimeric cytokines and their receptors).

The invention also relates to methods for modulating (as defined in WO09/068627) IL-23 and/or IL-23-mediated signaling (as defined in WO09/068627), either in vitro (e.g. in an in vitro or cellular assay) orin vivo (e.g. in an a single cell or multicellular organism, and inparticular in a mammal, and more in particular in a human being, such asin a human being that is at risk of or suffers from a disease ordisorder associated with IL-23 and/or its receptors), which methodcomprises at least the step of contacting IL-23 with an anti-IL-23polypeptide of the invention, in a manner and in an amount suitable tomodulate IL-23 and/or IL-23-mediated signaling.

The invention also relates to the use of an anti-IL-23 polypeptide ofthe invention in the preparation of a composition (such as, withoutlimitation, a pharmaceutical composition or preparation as furtherdescribed herein) for modulating (as defined in WO 09/068627) IL-23and/or IL-23-mediated signaling (as defined in WO 09/068627), either invitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an asingle cell or multicellular organism, and in particular in a mammal,and more in particular in a human being, such as in a human being thatis at risk of or suffers from a disease or disorder associated withIL-23 and/or IL-23 mediated signaling).

The invention also relates to an anti-IL-23 polypeptide of the invention(or a composition comprising the same) for use in modulating signalingthat is mediated by IL-23 and/or its receptor(s).

The invention also relates to an anti-IL-23 polypeptide of the invention(or a composition comprising the same) for use in the prevention ortreatment of diseases associated with IL-23 and/or with signaling thatis mediated by IL-23, such as for example inflammation and inflammatorydisorders such as bowel diseases (ulcerative colitis, Crohn's disease,IBD), infectious diseases, psoriasis, cancer, autoimmune diseases (suchas MS), sarcoidosis, transplant rejection, cystic fibrosis, asthma,chronic obstructive pulmonary disease, rheumatoid arthritis, viralinfection, common variable immunodeficiency.

The invention also relates to a method for preventing or treating adisease associated with IL-23 and/or with signaling that is mediated byIL-23, such as for example inflammation and inflammatory disorders suchas bowel diseases (ulcerative colitis, Crohn's disease, IBD), infectiousdiseases, psoriasis, cancer, autoimmune diseases (such as MS),sarcoidosis, transplant rejection, cystic fibrosis, asthma, chronicobstructive pulmonary disease, rheumatoid arthritis, viral infection,common variable immunodeficiency, said method comprising administeringto a subject in need of such treatment a prophylactic or therapeuticallyactive amount of an anti-IL-23 polypeptide of the invention (or of acomposition comprising the same).

The invention will now be further described by means of the followingnon-limiting preferred aspects, examples and figures, in which:

FIG. 1 lists the amino acid sequences referred to herein (SEQ IDNOs:1-23, see left column). The second column from left lists thereference SEQ ID NOs of the sequences if they appeared in previousapplications, i.e., in WO 2009/068627, WO 2010/142534, or WO2011/135026;

FIG. 2 is a table listing some of the amino acid positions that will bespecifically referred to herein and their numbering according to somealternative numbering systems (such as Aho and IMGT);

FIG. 3 shows an alignment of the amino acid sequences referred toherein.

FIG. 4 shows two corresponding plots of data points obtained in Example1 when 66 serum samples from human healthy subjects and 29 samples fromSLE patients were tested for binding to SEQ ID NO:1 (reference) and SEQID NOs: 2 and 3 (invention). Each dot represents the binding level forone of the 96 samples tested. The data points shown in the right handpanel and the left hand panel are the same; in the right hand panel thedata points measured with each individual sample for each of the threecompounds tested (i.e. SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3,respectively) are connected by means of a line (as a result, thedeclination of the line gives an indication of the extent to whichbinding by pre-existing antibodies is reduced when the mutations of theinvention and the C-terminal alanine are introduced);

FIG. 5 details the data for binding to SEQ ID NO:1 (reference) and SEQID NOs: 2 and 3 (invention) of pre-existing antibodies from 10representative samples from SLE patients tested in Example 1 and shownin FIG. 4.

FIG. 6 is a table listing the binding data (3 columns giving normalizedPre-existing antibody (“Pre-Ab”) binding levels (RU at 125) and 2columns giving percentage of reduction in Pre-existing antibody bindingcompared to SEQ ID NO:1, respectively) of the data points compiled inFIG. 4.

EXPERIMENTAL PART

The human samples used in the Experimental Part below were eitherobtained from commercial sources or from human volunteers (after allrequired consents and approvals were obtained) and were used inaccording with the applicable legal and regulatory requirements(including but not limited to those regarding medical secret and patientprivacy)

In the Examples below, unless explicitly indicated otherwise, thebinding of pre-existing antibodies that are present in the samples used(i.e. from healthy volunteers, and SLE patients) to the NANOBODIES®tested was determined using PROTEON™ (protein interaction analysissystem) as follows:

NANOBODIES® were captured on human serum albumin and the binding ofpre-existing antibodies on the captured NANOBODIES® was evaluated usingthe PROTEON™ XPR36 (Bio-Rad Laboratories, Inc.). PBS/TWEEN®(polyethylene glycol sorbitan monolaurate) (phosphate buffered saline,pH7.4, 0.005% TWEEN®20) was used as running buffer and the experimentswere performed at 25° C. The ligand lanes of a PROTEON™ GLC Sensor Chipwere activated with EDC/NHS (flow rate 30 μl/min) and HSA was injectedat 10 μg/ml in PROTEON™ Acetate buffer pH4.5 (flow rate 100 μl/min) torender immobilization levels of approximately 3200 RU. Afterimmobilization, surfaces were deactivated with ethanolamine HCl (flowrate 30 μl/min). NANOBODIES® were injected for 2 minutes at 45 μl/minover the HSA surface to render a NANOBODY® capture level ofapproximately 600 RU. The samples containing pre-existing antibodieswere centrifuged for 2 minutes at 14,000 rpm and supernatant was diluted1:10 in PBS-TWEEN®20 (0.005%) before being injected for 2 minutes at 45μl/min followed by a subsequent 400 seconds dissociation step. Aftereach cycle (i.e. before a new NANOBODY® capture and blood sampleinjection step) the HSA surfaces were regenerated with a 2 minuteinjection of HCl (100 mM) at 450/min. Sensorgram processing and dataanalysis was performed with PROTEON™ Manager 3.1.0 (Bio-RadLaboratories, Inc.). Sensorgrams showing pre-existing antibody bindingwere obtained after double referencing by subtracting 1) NANOBODY®-HSAdissociation and 2) non-specific binding to reference ligand lane.Binding levels of pre-existing antibodies were determined by settingreport points at 125 seconds (5 seconds after end of association).Percentage reduction in pre-existing antibody binding was calculatedrelative to the binding levels at 125 seconds of a reference NANOBODY®.

Example 1: Binding of Pre-Existing Antibodies to SEQ ID NO:1(Reference), SEQ ID NO:2 (Invention) and SEQ ID NO:3 (Invention)

The amino acid sequences of SEQ ID NO:1 (reference), SEQ ID NO:2(invention) and SEQ ID NO:3 (invention) were tested for binding bypre-existing antibodies present in serum samples obtained from 66healthy human volunteers and 29 SLE patients using PROTEON™ using theprotocol described in the preamble to the Experimental Part. Thesequences tested were captured using human serum albumin.

The results are shown in FIG. 4. FIG. 5 details the results obtained for12 representative SLE samples (i.e. taken from the 29 tested SLEsamples). FIG. 6 gives the data shown in FIG. 4.

The results show that the compounds of SEQ ID NOs: 2 and 3 show muchreduced binding by pre-existing antibodies compared to the compound ofSEQ ID NO:1 in a large number of samples obtained from healthy humanvolunteers as well as samples from SLE patients.

The invention claimed is:
 1. Polypeptide comprising an amino acid sequence that is chosen from the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO:
 3. 2. Polypeptide comprising the amino acid sequence of SEQ ID NO:
 2. 3. Polypeptide comprising the amino acid sequence of SEQ ID NO:
 3. 4. Composition comprising a polypeptide according to claim
 1. 5. A method for treating colitis, Crohn's disease, inflammatory bowel disease, psoriasis, or rheumatoid arthritis, comprising administering to a subject in need of such treatment a therapeutically active amount of a polypeptide according to claim
 1. 6. A method for treating colitis, Crohn's disease, inflammatory bowel disease, psoriasis, or rheumatoid arthritis, comprising administering to a subject in need of such treatment a therapeutically active amount of a polypeptide according to claim
 2. 7. A method for treating colitis, Crohn's disease, inflammatory bowel disease, psoriasis, or rheumatoid arthritis, comprising administering to a subject in need of such treatment a therapeutically active amount of a polypeptide according to claim
 3. 8. A method for treating colitis, Crohn's disease, inflammatory bowel disease, psoriasis, or rheumatoid arthritis, comprising administering to a subject in need of such treatment a therapeutically active amount of a composition according to claim
 4. 